1. Field of Invention
The present invention relates to methods for crosslinking hyaluronan solutions and nanofibrous membranes without the use of additional chemical cross-linking agent.
2. Discussion of the Background
Hyaluronan (HA) is an associated polymer, having the following structure:

HA has an acidic group as well as a glucosamine segment. As shown above, HA is a linear polysaccharide consisting of alternating 1,4-linked units of 1,3-linked glucoronic acid and N-acetylglucosamine. The presence of the weak acid group makes the polymer a polyelectrolyte, i.e., its charge density depends on the degree of dissociation, that can be influenced by factors including, but not limited to:                pH        ionic strength        nature of co-ions and counter ions        solvent quality that shall also affect the above 3 conditions.        
The degree of association can be disturbed by physical and/or chemical means. For example:                By physical means, e.g., ultra-sonics, shear, microwave, etc.        By chemical means, such as complex formation with a liquid, e.g., polyethylene oxide is soluble in water because of its hydrogen bonding with water.        
The occurrence of HA is widespread in animals and bacteria, particularly in synovial fluid, vitreous humor, rooster comb, and the umbilical cord. HA is a polyanionic acid that has unique physicochemical properties and distinctive biological functions. Its unusually high viscosity has been utilized for surgical treatments in ophthalmology as a viscoelastic biomaterial protecting ocular cells from damage during the surgery.1-7 The lubricious polysaccharide is also used in orthopedics for the treatment of osteoarthritis of the knee through injection of the aqueous solution.8-11 In addition, HA is applied in cosmetics to prevent the skin from drying because of its high retention ability12 and in drug delivery systems because of its biodegradability.13-16 
Although HA can be produced on a large scale by biotechnology,17 applications of HA are still limited because the hydrophilic HA can be readily dissolved in water and undergoes relatively rapid biodegradation in vivo. Many attempts have been made to introduce cross-linking sites into HA molecules to produce insoluble or gel-like HA materials. The cross-linking reagents used in previous studies included diopeoxy,18 glutaraldehyde,19 carbodiimide20,21 and disulfide.22 
Unfortunately, conventional chemical modifications and subsequent cross-linking of HA have an inevitable problem of extra risks, such as toxicity and bio-incompatibility intrinsic to most chemical modifications. However, one method of producing HA gels by freezing and thawing acidic aqueous solution of HA has been proposed in a US patent (U.S. Pat. No. 6,387,413B1)23. The obtained HA gel kept its shape for at least one day in a neutral aqueous solution at 25° C.
Previously, the present inventors successfully prepared electrospun HA nanofiber membranes by a blowing-assisted electro-spinning method.24 However, such electro-spun HA nano-fibrous membranes, due to the very large surface-to-volume ratios, dissolve instantly in water. Accordingly, a pathway is needed to fabricate hydrophilic, but essentially water-resistant, electro-spun HA membranes without chemical modifications.